EP1743178A2 - Nachweisverfahren für einen liganden der selektiv eine funktionelle kaskade modulieren kann und ein target impliziert sowie dessen verwendung für hochdurchsatz-screening von molekülen die von interesse sind - Google Patents
Nachweisverfahren für einen liganden der selektiv eine funktionelle kaskade modulieren kann und ein target impliziert sowie dessen verwendung für hochdurchsatz-screening von molekülen die von interesse sindInfo
- Publication number
- EP1743178A2 EP1743178A2 EP05762419A EP05762419A EP1743178A2 EP 1743178 A2 EP1743178 A2 EP 1743178A2 EP 05762419 A EP05762419 A EP 05762419A EP 05762419 A EP05762419 A EP 05762419A EP 1743178 A2 EP1743178 A2 EP 1743178A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- target
- antibody
- molecules
- fragment
- modulating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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Classifications
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to a method for identifying a ligand capable of selectively modulating a functional cascade involving a target or target molecule, as well as to its applications for high-throughput screening of molecules of interest, in particular therapeutic (drug).
- the discovery of new drugs involves developing methods of high-throughput screening of libraries of molecules that are effective, that is to say allowing to isolate from a limited number of selected molecules ( keys or hits), molecules active in vivo on the pathology that we are trying to treat (leads or leads).
- the functional tests in vitro in cell culture or in vivo in an appropriate animal model
- the isolated molecules must be able to specifically modulate the property of interest of the target without affecting its other activities whose modulation could have deleterious effects for the cell.
- screening must be highly specific in order to limit as much as possible the number of hits identified, so as to have only to test a small number in functional tests in vivo.
- screening methods must also be easy to implement and applicable to any target.
- screening methods based on the identification of molecules (mimotopes) mimicking the interaction between the target and a peptide or an oligonucleotide (aptamer) have been proposed. Screening methods using aptamers and / or peptides are illustrated in Lapan et al., Expert Opin. Ther. Targets, 2002, 6, 507-516; Green et al., BioTechniques, 2001, 30, 1094-1110; Burgstaller et al., Drug Discovery Today,
- aptamers / peptides capable of binding to the target are selected in vitro and then their modulating activity, with respect to the target, is verified by a functional test in vitro in an appropriate cell system or in vivo in an appropriate animal model.
- the aptamers / peptides having modulating activity on the target are used in competitive binding tests to identify hits capable of displacing the existing bond between the aptamer / peptide and the target.
- the modulating activity of the keys vis-à-vis the target is verified by a functional test in vitro in cell culture or in vivo in an appropriate animal model, so as to isolate molecules "heads of series" (leads) .
- targets such as the platelet-derived human growth factor isoform BB (PDGF BB) and the human immunodeficiency virus (NIH) Rev protein.
- PDGF BB platelet-derived human growth factor isoform BB
- NASH human immunodeficiency virus
- Antibodies have the advantage of recognizing any region of a protein, specifically and with a high affinity (of the order of nM).
- the inventors screened a library of 3000 molecules using an antibody directed against the SH2 domains of the protein tyrosine kinase Syk and capable of inhibiting the PLC ⁇ -2 pathway; among the 10 ligands of the Syk protein which they selected, at least one was an inhibitor molecule in vivo, acting on the path of PLC- ⁇ 2, without altering that of Ras also dependent on the Syk protein.
- aptamers and peptides are capable of recognizing any epitope on the surface of an antigen.
- peptides and aptamers are more suitable for interacting in the target cavities rather than on the surface, which is favorable for the inhibition of an enzymatic activity but not very interesting for inhibiting functional cascades.
- a number of recombinant monoclonal antibodies are used in therapeutic treatments.
- cetuximab anti-EGFR
- rituximab anti-CD20
- infliximab anti-TNF
- the screening process proposed by the inventors makes it possible to replace these antibodies with chemical molecules having the same inhibitory properties, which are easier to produce at a very much lower cost (by at least a factor of 10).
- the subject of the present invention is therefore a method of identifying a ligand capable of selectively modulating a functional cascade involving a target, characterized in that it comprises at least the following steps: a) identifying a antibody or an antibody fragment comprising at least one of the variable domains of an immunoglobulin chain, capable of binding to said target and of modulating said functional cascade involving said target, b) screening, from a library of molecules, ligands which modulate the binding between said target and the antibody or the antibody fragment identified in a), and c) identification from the modulating ligands obtained in b), of those capable of modulating said functional cascade.
- a positive or negative modulation activation or inhibition, total or partial, • "target involved in a functional cascade", any molecule of a cell of an organism (human, animal, plant) or of a microorganism (virus, bacteria , fungus, parasite) which is capable of interacting with one or more ligands (or natural partners, known or unknown), each binding to a site or region distinct from said target (FIG. 2); the binding of the ligand modulates a functional cascade involved in a physiological or pathological process of said cell or of said microorganism and results in a measurable biological activity in said cell or said microorganism.
- target involved in a functional cascade any molecule of a cell of an organism (human, animal, plant) or of a microorganism (virus, bacteria , fungus, parasite) which is capable of interacting with one or more ligands (or natural partners, known or unknown), each binding to a site or region distinct from said target (FIG. 2); the binding of the ligand
- Said target can be in particular: a protein, a peptide, a polynucleotide (DNA, RNA), a lipid, a sugar or a derivative of the above (glycoprotein, glycolipid ).
- FIG. 3 illustrates the example of the protein tyrosine kinase (PTK) Syk which is a multifunctional protein involved inter alia in two activation cascades: the first (cascade 1) which leads to the activation of the Ras protein then of the MAPK, is involved in the growth and differentiation of certain cells of the lymphoid line including B cells, mast cells, basophils and a subpopulation of T cells (first biological activity), and the second (cascade 2) which leads to activation of PTK Btk, phospholipase C- ⁇ 2 (PLC ⁇ 2) and increased intracellular calcium flow, results in degranulation and the release of allergic mediators by mast cells (second biological activity).
- PTK protein tyrosine kinase
- cascade 2 which has applications in the prevention and / or treatment of allergies, while cascade 1 should not be inhibited as it could result in harmful side effects for cells (tumor aggressiveness; Coopman et al., Nature, 2000, 406, 742-747).
- molecule bank or chemistry library.
- a set of molecules other than antibodies or fragments of antibodies related by their structure, their origin or their function, in particular a combinatorial library including molecules which differ from one another by the systematic or random replacement of their elementary constituents, for example a bank of oligomers such as peptides, oligonucleotides (aptameres) and oligosaccharides or else a bank of organic molecules other than oligomers, cyclic or non-cyclic, in particular small organic molecules, that is to say of mass molecular less than 2500 Da, preferably less than 2000 Da, more preferably less than 1500 Da, more preferably less than 1000 Da, even more preferably less than 750 Da.
- oligomers such as peptides, oligonucleotides (aptameres) and oligosaccharides
- organic molecules other than oligomers cyclic or non-cyclic, in particular small organic molecules, that is to say of mass molecular less than 2500 Da, preferably less than 2000 Da, more
- antibody fragment a fragment capable of binding to a target, comprising at least the variable domain of a heavy chain (NH) and / or the variable domain of a light chain (NL) of an immunoglobulin standard, or the variable domain of a single chain immunoglobulin, such as the Fab, Fv, scFv or NHH fragments.
- antibody or antibody fragment capable of binding to said target and of modulating a functional cascade involving said target an antibody or an antibody fragment capable of binding to said target and of mimicking the in vivo interaction of said target with its ligand (natural partner), at the site of interest; the target epitope recognized by said antibody or antibody fragment is partially or totally overlapping with the site of interest of said target (FIG. 2B).
- step b) The screening of the bank of molecules [step b)] or of a bank of antibodies or of antibody fragments [step a) of identification of the antibodies or antibody fragments capable of binding to the target] are produced by any method known to those skilled in the art allowing the measurement of the interaction between two partners (ELISA, energy transfer by resonance (FRET), fluorescence polarization , surface plasmon resonance).
- Steps a) and b) are carried out using a target or a derivative of the target such as a fragment comprising at least the site of interest, a mimotope, or alternatively an anti-idiotypic antibody representing the internal image of said target site of interest.
- Said target and its derivatives or the antibody or the antibody fragment are optionally immobilized on an appropriate support, and / or labeled by any means allowing a measurable signal to be obtained, known to those skilled in the art.
- the identification of ligands or of antibodies or antibody fragments capable of selectively modulating a functional cascade involving said target is carried out by a functional test making it possible to measure the biological activity resulting from said functional cascade. It is, in particular an in vitro test, in an appropriate cellular system or in vivo, in an appropriate organism.
- the cells in culture can be immortalized or in primary culture, adherent or in suspension.
- the living organism can be any organism or microorganism of laboratory or study (animal (rodents, rabbits, pigs, cattle, sheep ...) or transgenic or non-transgenic plant, parasite, ..).
- animal rodents, rabbits, pigs, cattle, sheep
- transgenic or non-transgenic plant parasite, ..
- the cellular systems and appropriate organisms there may be mentioned in particular those which are models of the physiological or pathological process involving said functional cascade, such as, without limitation, models of diabetes, allergy, arthritis or asthma.
- the identification of antibodies or antibody fragments capable of modulating a functional cascade involving said target is carried out in cells modified by an expression vector for said antibodies or their fragments (antibodies or fragments of intracellular antibodies).
- the functional test involved in this verification in vitro, in a cellular system or in vivo in an appropriate organism depends on the biological activity which one seeks to modulate for a therapeutic or non-therapeutic aim.
- biological activity measurable in cells there may be mentioned in particular: division, migration, degranulation, transport to across membranes, differentiation, apoptosis, replication, transcription and the production of factors such as cytokines.
- the functional tests making it possible to measure said biological activity in vitro or in vivo are known to a person skilled in the art.
- the identification of said antibody or antibody fragment in a) is carried out by screening a bank of antibodies or antibody fragments, preferably a bank of scFv fragments, preferably, a phage library expressing scFv fragments on their surface.
- the identification of said antibody can comprise: - a first step of screening for antibodies or antibody fragments capable of binding to said target, in particular in phage-ELISA and - a second step of identification of antibodies or antibody fragments capable of modulating said functional cascade, in particular in cells modified by an expression vector for said antibodies or antibody fragments (antibody or intracellular antibody fragments).
- the antibodies or the antibody fragments, in particular the scFv fragments selected during the screening step can then be cloned in an appropriate vector and expressed in cells, in particular eukaryotic cells such as mammalian cells modified by the recombinant vector thus obtained.
- eukaryotic cells such as mammalian cells modified by the recombinant vector thus obtained.
- Such antibodies or fragments of antibodies called intracellular antibodies (Cattaneo et al., Trends in Biotechnology, 1999, 17, 115-121), are expressed in functional form, capable of binding the target in the compartment of the modified cells in which it is expressed (nucleus, cytoplasm, secretory compartment).
- mutations can be introduced into the sequence of antibodies or selected antibody fragments, so as to improve their solubility and / or their conformation in cells, in particular in eukaryotes.
- mutations that may be envisaged, mention may be made in particular of the point mutations described in Hurle et al., PNAS USA, 1994, 91, 5446-5450 and Martineau et al., J. Mol. Biol., 1998, 280, 117-127.
- antibodies or antibody fragments with increased stability and / or affinity can be selected by a additional phenotypic selection step, as described in Cattaneo et al., cited above.
- step b) of screening comprises: contacting the bank of molecules to be tested with the target or one of its derivatives as defined above, - the addition of the antibody or of the fragment of antibody, optionally labeled, and - the measurement, by any appropriate means, of the relative quantity of antibody bound to the target, in the presence or in the absence of said library of molecules.
- step b) of screening is carried out by a binding test of said target, in competition with said antibody or said antibody fragment, so as to isolate ligands from said target which displace said antibody or antibody fragment from the target / antibody complex or antibody fragment.
- An example of this embodiment includes: - bringing the antibody or antibody fragment, optionally labeled, into contact with the target or one of its derivatives as defined above, - addition of the library of molecules to be tested so as to detect the molecules capable of displacing the antibody or the antibody fragment from its complex with the target fragment, and - measuring, by any suitable means, the relative quantity of antibody bound to the target, in the presence or in the absence of said library of molecules.
- Another example of this mode of implementation comprises: the mixing of the antibody or of the antibody fragment, optionally labeled, with the library of molecules to be tested, - bringing the mixture into contact with the target or one of its derivatives as defined above, and - measuring, by any suitable means, the relative amount of antibody bound to the target, in the presence or in the absence of said bank of molecules.
- the capacity of a molecule to modulate the binding between the target and the antibody is determined by comparing the value of the binding signal of the antibody, in the presence or in the absence of each of the molecules of the library, tested separately.
- steps a) and c) are carried out by a functional test making it possible to measure the biological activity resulting from said functional cascade, in particular by an in vitro test, in an appropriate cell system or in vivo, in an appropriate organism.
- said target is selected from: an enzyme, a receptor, an adapter protein, a transporter, a chaperone protein, a regulatory protein.
- enzymes such as tyrosine kinase proteins such as the Syk protein; cytokine receptors such as the IL-13, EGF or TNF receptor, adapter proteins such as SLP-76 and E6-AP; chaperone proteins such as HSP70 and HSP60; regulatory proteins such as NF- ⁇ B, I- ⁇ B, Akt, PSAT-1, p53, p73 and the Bcl2 family.
- said antibody or antibody fragment in a) is directed against the SH2 domains of the Syk protein and it is capable of inhibiting the PLC ⁇ -2 pathway.
- said bank of molecules in b) is a bank of small organic molecules.
- the present invention also relates to a kit for implementing the method as defined above, characterized in that it comprises at least: a target involved in a functional cascade, a fragment of said target comprising at least the site of interest, a mimotope of said target, or alternatively an anti-idiotypic antibody representing the internal image of said site of interest of the target as defined above, - an antibody or an antibody fragment comprising at least one of the variable domains of an immunoglobulin chain, capable of binding to said target and of modulating said functional cascade involving said target or a bank of antibodies or antibody fragments, as defined above, and - a bank of molecules to be tested, as defined above.
- the method according to the invention can be used to screen any type of molecule of therapeutic or non-therapeutic interest, capable of acting on a physiological or pathological process of any type of cell or microorganism such as defined above. It can be used to screen new families of molecules with new properties of therapeutic or non-therapeutic interest or derivatives of these molecules with specific activity and / or improved therapeutic index.
- the banks of molecules are prepared according to conventional methods of combinatorial chemical synthesis, known to those skilled in the art.
- the antibody or the antibody fragment as defined above are prepared by standard techniques known to those skilled in the art, such as those described in Antibodies: A Laboratory Manual, E. Howell and D Lane, Cold Spring Harbor Laboratory, 1988.
- the monoclonal antibodies are produced from hybridomas obtained by fusion between B lymphocytes of an immunized animal and myelomas, according to the technique of Kohler and Milstein (Nature, 1975, 256, 495-497); the hybridomas are cultivated in vitro, in particular in fermenters or produced in vivo, in the form of ascites; alternatively, said monoclonal antibodies are produced by genetic engineering as described in Methods in Molecular Biology: Antibody Phage Display Methods and Protocols, P.M. O'Brien and R. Aitken. Humana Press, Vol. 178, 2002.
- suitable animals are repeatedly immunized with the target or one of its fragments, according to a standard protocol comprising a first immunization by intraperitoneal injection of the antigen in an equivalent volume of complete Freund's adjuvant then a second immunization (booster) 15 days later under identical conditions but this time with incomplete Freund's adjuvant.
- the monoclonal antibodies are produced according to a standard protocol comprising the sacrifice of the animals two weeks after the last booster, the removal of the spleen, the suspension of the spleen lymphocytes and the fusion of these lymphocytes with the murine cell line SP2 / 0 which does not produces no murine antibody, which is immortalized, and has all the machinery necessary for the secretion of immunoglobulins.
- the antibody banks are either natural banks produced from the V regions H and N
- the specific antibodies of the target are isolated by screening of the banks as defined above, in particular of the phage banks; after several selection steps, the phages which express the target-specific antibody fragments are isolated and the cDNAs corresponding to said fragments are expressed in an appropriate expression system, by conventional techniques of cloning and DNA expression recombinant.
- said cDNAs are cloned both into a prokaryotic and eukaryotic expression vector, in the form of a fusion protein comprising at least at its end -NH 2 or COOH, a label for the detection of said antibody fragment, for example an epitope (c-myc, HA).
- Said fusion protein may optionally include, at one end, a label for the purification of the antibody fragment, for example a polyhistidine sequence for purification on a nickel-agarose column.
- Said eukaryotic expression vector contains, in addition to the appropriate transcription / translation regulatory elements (promoter, enhancer, Kozak consensus sequence, polyadenylation signal .7) under the control of which are inserted the coding sequences as defined above, the elements essential for the expression of said fragments in the appropriate cell compartment as defined above, in particular in the cytoplasm, and optionally a selection marker (gene for resistance to an antibiotic, etc.) for the selection of cell lines, in particular eukaryotes, modified in a stable manner by said expression vector and producing intracellular antibodies.
- the monoclonal antibodies or their fragments as defined above are purified by conventional techniques known to those skilled in the art, in particular by affinity chromatography.
- the target or the fragment comprising the site of interest can be either purified from tissues or cells, by standard techniques known to those skilled in the art, or produced by standard recombinant DNA techniques known from A person skilled in the art, following standard protocols such as those described in particular in Current Protocols in Molecular Biology (Frederick M. A USUBEL, 2000, Wiley and son Inc, Library of Congress, USA).
- the polynucleotide encoding said protein or said fragment is cloned into an expression vector (plasmid, virus, etc.) in which said polynucleotide is placed under the control of regulatory elements for appropriate transcription and translation.
- said vector can comprise sequences (labels or tags) fused in phase with the 5 ′ and / or 3 ′ end of said polynucleotide, useful for the immobilization, and / or the detection and / or purification of the protein.
- expressed from said vector said protein is then expressed in appropriate host cells (bacteria, yeasts, insect or mammalian cells) and optionally purified.
- appropriate host cells bacteria, yeasts, insect or mammalian cells
- the fragment comprising the site of interest is synthesized in solid phase, according to the method originally described by Merrifield et al. (J Am. Chem. Soc, 1964, 85: 2149-).
- the labeling of the target, of the antibody or of its fragment and the detection of the antibody / target and ligand / target bond are carried out by the conventional techniques known to those skilled in the art: (i) by fusion of the sequence coding with the sequence of an epitope (c-myc, HA), (ii) by coupling with an appropriate marker such as a fluorophore, an enzyme (alkaline phosphatase, peroxidase), an isotope radioactive, or biotin, (iii) by a labeled secondary antibody or by any specific interaction with an appropriate marker.
- an appropriate marker such as a fluorophore, an enzyme (alkaline phosphatase, peroxidase), an isotope radioactive, or biotin
- the antibody / target and ligand / target bond is detected by fluorescence transfer between two complementary fluorescent proteins (Philipps et al., J.
- two complementary fluorophores fluorescein / teramethylrhodamine
- said target or said fragment comprise an acceptor or donor fluorophore respectively at their NH end 2 and / or COOH
- the antibody or the antibody fragment comprise the complementary fluorophore at their NH and / or COOH end.
- the method according to the invention has the following advantages: • it makes it possible to identify new molecules which act selectively and specifically on a pathophysiological process, potentially usable as a drug, by the combination of two screening steps: - a screening step by a binding test, easily automatable and adaptable to broadband, molecules which mimic the interaction of a target with an antibody or an antibody fragment (idiotypic approach) and are therefore capable of modulating the functional cascade involving said target, and - a step of low-throughput screening, by a functional test in cell culture or in an appropriate animal model, of molecules capable of modulating the functional cascade involving said target, in particular by measuring the biological activity resulting from said cascade.
- the results obtained from a library of 3000 molecules show that the efficiency of the first step of screening with the anti-Syk antibody is such that the second step of functional screening on a limited number of molecules (10 molecules), makes it possible to isolate at least one inhibitory molecule active in vivo on the selected functional cascade.
- the invention also comprises other arrangements which will emerge from the description which follows, which refers to examples of screening for molecules capable of selectively modulating the degranulation of mast cells, using a scFv fragment directed against the SH2 domains of the Syk target, of the PTK family (protein tyrosine kinase), as well as in the appended drawings in which: - Figure 1 is a schematic representation of the process according to the invention, - Figure 2 is a schematic representation of a target and the functional cascade in which it is involved, - FIG. 3 is a schematic representation of the activation cascades involving the protein tyrosine kinase Syk and the resulting biological activities, - FIG.
- FIG. 4 illustrates validation of the method according to the invention using the Syk protein as target and the scFv G4G11 fragment as antibody for the screening of a chemo library.
- the ten molecules resulting from the ELISA screening at least one of them strongly inhibits the release of inflammation mediators in the beta-hexosaminidase release test: at the concentration of 3 ⁇ M the drug inhibits 93% the degranulation induced by IgE but not that induced by ionomycin, - Figure 5 illustrates the comparison of the biological effects of the drug with the inhibition observed with scFv.
- Lysates of non-activated (NA) and activated (A) RBL-2H3 cells in the presence of the molecule selected in ELISA (drug) at a final concentration of 1.5 to 12 ⁇ M (a and b) or 3 ⁇ M ( c and d) or in the absence of this molecule, were analyzed by: a and b: immunoblot using an anti-phosphotyrosine 4G10 antibody (a), an anti-phosphoMAPK antibody (bl) or an anti-MAPK antibody (b2); c and d: immunoprecipitation using an anti-Syk (c) or anti-PLC- ⁇ 2 (d) antibody then immunoblot using the same antibody used as control, or the anti-phosphotyrosine 4G10 antibody .
- the Syk protein is a multifunctional protein tyrosine kinase which plays an important role in the activation of certain cells of the lymphoid line, including B lymphocytes, mast cells and basophils, and a subpopulation of T lymphocytes.
- the mast cell line following recognition by the IgE receptor (Fc ⁇ RI), of IgE associated with an allergen, results in the release of allergic mediators such as histamine and serotonin.
- the aggregation of the Fc ⁇ RI receptor rapidly leads to the phosphorylation of the Syk protein and its activation.
- Syk thus activated in turn phosphorylates its cytoplasmic substrates including the protein tyrosine kinase Btk and phospholipase C- ⁇ 2 (PLC- ⁇ 2), both involved in the activation cascade which results in the degranulation of mast cells and the release of allergic mediators ( PLC ⁇ -2 pathway; Figure 3).
- the Syk protein intervenes in a second path (FIG. 3) which leads to the activation of the Ras protein and then of the MAP kinase (MAPK).
- the secondary antibody is the monoclonal antibody 9E10 directed against the c-myc epitope (EQKLISEEDLN), coupled to peroxidase (Munro et al., Cell, 1986, 46, 291-300).
- the bank of molecules to be tested is a bank of 3000 small organic molecules (ChemBridge Corporation; http://chembridge.com). All dilutions and incubations were carried out in 0.1% PBS / Tween 20 / 0.1% BSA buffer. The washes were carried out in a 0.1% PBS / Tween 20 buffer.
- the substrate is tetramethylbenzidine dihydrochloride 0.1 mg / ml in a phosphate citrate buffer pH 5, supplemented with H O 2 (0.03%).
- the scFv fragment was then added at a concentration of 150 nM in a volume of 50 ⁇ l and the plates were incubated for 1 hour at room temperature. After a new wash, 50 ⁇ l of secondary antibody labeled with peroxidase were added and the plates were incubated for 30 minutes at room temperature. After a final wash, the peroxidase reaction substrate was added in a volume of 100 ⁇ l, then the plates were incubated for 5 minutes and the reaction was stopped by adding 50 ⁇ l of H 2 SO 4 2N. The optical density at 450 mm was then measured using an automatic reader for ELISA plate.
- the RBL-2H3 cells (basophilic rat leukemia line) are seeded in a 96-well plate at the rate of 2.10 5 cells / well. They are sensitized in the presence of an anti-DNP IgE monoclonal antibody, overnight at 37 ° C. The cells are then incubated for 2 hours at 37 ° C., in the presence of different concentrations of the molecule selected by ELISA, then they are activated by the DNP-BSA antigen, 3 minutes at 37 ° C. The culture supernatant is then harvested and stored at 4 ° C (SI). Adherent cells are lysed in the presence of a lysis buffer containing 0.1% Triton X-100 and protease inhibitors.
- Cell lysates are also harvested and stored at 4 ° C (S2).
- S2 the solutions S1 and S2 are incubated with the substrate of the enzyme: 4-nitrophenyl-2-deoxy- ⁇ -D-glucopyranoside, for 1 h 30 at 37 ° C.
- the enzymatic reaction is stopped by adding a glycine solution pH 3 and the optical density is read at 405 nm.
- the quantity of ⁇ -hexosaminidase released corresponds to: Hex Sl x 100 (Hex Sl + Hex S2)
- a degranulation test with a calcium ionophore (ionomycin) was carried out as a control.
- mice Verification of the modulating power of the selected molecules, in vivo, in mice.
- mice BALB / c mice receiving no drugs and making it possible to measure the maximum anaphylactic shock (positive control).
- the effect of the molecule on anaphylactic shock was then determined by measuring the release of Evans blue, by extravasation. To do this, the mice were sacrificed, then their ears were cut off. Equal surfaces of the ears were removed, using a cookie cutter, then they were chopped, and put in a solution of formamide, overnight at 55 ° C. The following day, the release of Evans blue in the formamide solution was determined by measuring the optical density at 610 nm. The results show a 70% reduction in the extravasation of Evans blue in the mice having received the active molecule in the mast cell degranulation inhibition test (FIG. 4), compared to the control mice receiving no molecule. or an inactive molecule. These results demonstrate the capacity of the molecule selected by the screening method according to the invention, to protect an animal from anaphylactic shock.
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Application Number | Priority Date | Filing Date | Title |
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FR0404433A FR2869416B1 (fr) | 2004-04-27 | 2004-04-27 | Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible et ses applications pour le criblage a haut-debit de molecules d'interet. |
PCT/FR2005/001020 WO2005106481A2 (fr) | 2004-04-27 | 2005-04-25 | Procede d'identification d'un ligand capable de moduler selectivement une cascade fonctionnelle impliquant une cible, et ses applications pour le criblage a haut-debit de molecules d'interet. |
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EP1743178A2 true EP1743178A2 (de) | 2007-01-17 |
EP1743178B1 EP1743178B1 (de) | 2010-06-16 |
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US (1) | US9110077B2 (de) |
EP (1) | EP1743178B1 (de) |
JP (1) | JP4814220B2 (de) |
AT (1) | ATE471518T1 (de) |
CA (1) | CA2564767C (de) |
DE (1) | DE602005021875D1 (de) |
ES (1) | ES2348478T3 (de) |
FR (1) | FR2869416B1 (de) |
WO (1) | WO2005106481A2 (de) |
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US20090309617A1 (en) * | 2007-08-24 | 2009-12-17 | Ye Fang | Biosensor antibody functional mapping |
FR2929851B1 (fr) | 2008-04-09 | 2012-11-30 | Centre Nat Rech Scient | Molecules inhibant une voie metabolique impliquant la proteine tyrosine kinase syk et procede d'identification de ces molecules |
EP2752487A1 (de) | 2013-01-03 | 2014-07-09 | Sanofi | Intrazelluläres Phänotypen-Screening |
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US5858657A (en) * | 1992-05-15 | 1999-01-12 | Medical Research Council | Methods for producing members of specific binding pairs |
EP0676966A4 (de) * | 1992-12-31 | 1997-01-29 | Univ Ramot | Antikörper gerichtet gegen durch bindung assoziierte epitope. |
JPH08509380A (ja) * | 1993-04-23 | 1996-10-08 | アリアド・ファーマシューティカルズ・インコーポレイテッド | ヒトSyk |
US6010861A (en) * | 1994-08-03 | 2000-01-04 | Dgi Biotechnologies, Llc | Target specific screens and their use for discovering small organic molecular pharmacophores |
JP2002515744A (ja) * | 1996-05-20 | 2002-05-28 | シグナル ファーマシュウティカルズ,インコーポレイテッド | マイトジェン活性化プロテインキナーゼp38―2およびその使用方法 |
GB9726851D0 (en) * | 1997-12-19 | 1998-02-18 | Zeneca Ltd | Human signal transduction serine/threonine kinase |
GB2344886B (en) * | 1999-03-10 | 2000-11-01 | Medical Res Council | Selection of intracellular immunoglobulins |
AU5013200A (en) * | 1999-05-13 | 2000-12-05 | Caliper Technologies Corporation | Target independent assay systems and methods |
AU6058500A (en) * | 1999-06-30 | 2001-01-31 | Center For Blood Research, The | Fusion protein and uses thereof |
US6709839B1 (en) * | 1999-09-24 | 2004-03-23 | Rigel Pharmaceuticals, Inc. | SYK-UBP proteins, compositions and methods of use |
JP2003009899A (ja) * | 2001-05-28 | 2003-01-14 | New Industry Research Organization | 化合物のスクリーニング方法 |
WO2003059943A2 (en) * | 2002-01-18 | 2003-07-24 | Karo Bio Ab | Conformation-specific, protein kinase binding peptides and related methods and products |
US7244592B2 (en) * | 2002-03-07 | 2007-07-17 | Dyax Corp. | Ligand screening and discovery |
WO2004020619A1 (en) * | 2002-08-30 | 2004-03-11 | Ingenium Pharmaceuticals Ag | Modified phospholipase c-gamma-2, expression products, and non-human animal models comprising said genes, and therapeutic uses |
ITRM20020588A1 (it) * | 2002-11-21 | 2004-05-22 | Lay Line Genomics Spa | Metodo per isolare anticorpi intracellulari neutralizzanti di interazioni proteiche. |
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2004
- 2004-04-27 FR FR0404433A patent/FR2869416B1/fr not_active Expired - Fee Related
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- 2005-04-25 US US11/568,375 patent/US9110077B2/en not_active Expired - Fee Related
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- 2005-04-25 AT AT05762419T patent/ATE471518T1/de not_active IP Right Cessation
- 2005-04-25 WO PCT/FR2005/001020 patent/WO2005106481A2/fr active Application Filing
- 2005-04-25 DE DE602005021875T patent/DE602005021875D1/de active Active
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Also Published As
Publication number | Publication date |
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FR2869416A1 (fr) | 2005-10-28 |
US20080146453A1 (en) | 2008-06-19 |
EP1743178B1 (de) | 2010-06-16 |
JP2007534954A (ja) | 2007-11-29 |
US9110077B2 (en) | 2015-08-18 |
WO2005106481A2 (fr) | 2005-11-10 |
ES2348478T3 (es) | 2010-12-07 |
ATE471518T1 (de) | 2010-07-15 |
DE602005021875D1 (de) | 2010-07-29 |
CA2564767A1 (fr) | 2005-11-10 |
CA2564767C (fr) | 2014-06-17 |
WO2005106481A3 (fr) | 2006-11-02 |
FR2869416B1 (fr) | 2007-01-12 |
JP4814220B2 (ja) | 2011-11-16 |
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